Comunicación y medios Masivos

p

subunits.

4 . 1 I n t r o d u c t i o n

Characterisation of GABAa-R subunit phosphorylation in the cellular

environment requires specific purification of subunit polypeptides following metabolic labelling of intracellular ATP with and activation of specific protein kinases. Phosphorylated subunits may then be separated according to their size by SDS-PAGE and visualised by autoradiography. Biochemical subunit analysis allows correlation of specific phosphorylation events with specific effects on receptor function.

The preferred method for such biochemical analysis of protein phosphorylation involves the use of specific antibodies to purify proteins by immunoprécipitation. The use of this technique in G A B A a -R subunit purification has many advantages over purification by affinity chromatography. Most importantly, the use of antibodies allows purification of higher yields of receptor subunits from brain membranes or heterologous cell expression systems. Antibodies also precip itate specific subunits regardless of rec e p to r subunit combination, thereby allowing comparison of subunits without complications arising from receptor subtype diversity in neurons. Previous investigations using this approach have confirmed the identity of phosphorylation sites within p i and y2 subunits (Moss et al., 1992b; Krishek et al., 1994; Moss et al., 1995).

Large scale purification of G A B A a-R complexes by affinity chromatography on benzodiazepine affinity-columns initiated the cloning of the first cDNAs encoding receptor subunits (Sigel and Barnard, 1984; Schofield et al., 1987). Other receptor subunit cDNAs

were then cloned and their primary amino acid sequences have been described (Stephenson, 1995; Davies et al., 1997b). This information has allowed the design of subunit sequence specific peptides which have been used to raise a variety of subunit specific antibodies. Using peptides derived from N-terminal amino acid sequences, polyclonal and monoclonal antibodies have been raised which specifically recognise receptor a , P, y and 8 subunit isoforms (Schoch

et al., 1985; Sato and Neale, 1989; Stephenson et al., 1989; Benke et al., 1990; Stephenson et al., 1990; Buchstaller et al., 1991; Pollard et al., 1991; Endo and Olsen, 1992; Machu et al., 1993b).

Due to the high degree of sequence homology between p subunit isoforms, the development of p subunit-specific antibodies has proven difficult (Ymer et a/., 1989). The best described and most widely used anti-p subunit antibody is bdl7, a monoclonal antibody which was raised against purified G A B A a-R subunits. Epitope mapping has shown that bd l7 recognises the N-terminal 15 amino acid residues of the p2 subunit (Ewert et al., 1990), an epitope common to both p2 and p3 subunits. bd l7 is therefore unsuitable for studies requiring differentiation of these two p subunits.

To circumvent the problems of multiple subunit specificity, sequence specific peptides from p subunit intracellular domains have recently been used to produce polyclonal antibodies which recognise receptor p subunits (Endo and Olsen, 1992; Machu et al., 1993b; Benke et al.,

1994). These antibodies have allowed identification of individual receptor p subunits in neurons by immunoprécipitation, western blotting and immunofluorescence.

A previous report of GABAa-R p l subunit phosphorylation described production of an antibody specific for this subunit (Moss et al.,

1992b). This polyclonal antibody was raised by immunisation of 101

rabbits with the intracellular domain of the (31 subunit expressed and purified as a GST-fusion protein from E. coli (Moss et al., 1992a). Subunit specific antibodies affinity-purified from immune serum were shown to im m unoprecipitate p 1 subunit p o ly p ep tid es specifically from HEK293 cells after metabolic labelling. Use of these antibodies was succesful in the characterisation of P1 subunit phosphorylation by PKA and PKC in HEK293 cells (Moss et al., 1992b; Krishek et al., 1994).

In order to produce antibodies specific for GABAa-R P subunit isoforms it was decided to raise subunit specific polyclonal antibodies directed against the intracellular domains of GABAa-R P subunits

expressed as GST-fusion proteins. Polyclonal antibodies raised against such a large immunogen do not depend on recognition of a single epitope for immunoprécipitation and therefore are ideal for the study of receptor subunit phosphorylation which may alter individual intracellular epitopes. The production of such antibodies would allow study of recombinant receptor phosphorylation in HEK293 cells and could also facilitate immunoprécipitation of receptor subunits from neuronal preparations and whole brain lysates. Rabbits were immunised with GST-fusion proteins containing the intracellular domains of receptor p l, P2 and p3 subunits. Immune serum was screened for the ability to recognise receptor domains expressed as GST-fusion proteins and specific antibodies were affinity purified. Subunit specificity of purified antibodies was then exam ined by w estern b lo ttin g , im m u n o -flu o re s c e n c e and immunoprécipitation of full length receptor subunits expressed in HEK293 cells.

4 . 2 R abbit im munisation with fusion proteins containing intracellular domains of G A B A a-R

P

GST-fusion proteins containing intracellular domains of receptor p 1, P2 and p3 subunits were expressed and purified from E. coli as described (Moss et a l , 1992a; Figure 2). These proteins purified to near homogeneity, were used as antigens for immunisation of rabbits and production of polyclonal antisera. Prior to immunisation serum sa m p les w ere ta k en (p re b le e d ) to e s ta b li s h b a s e lin e immunoreactivity levels. Primary immunisations were carried out by subcutaneous injection of protein (lOOpg) at numerous sites in Freund's complete adjuvant. Boost immunisations of 50pg protein in Freund's incomplete adjuvant were administered 14, 21 and 49 days post immunisation. Test serum samples were obtained at days 35 and 56 post immunisation to allow monitoring of immune responses (Figure 15).

G S T - P 1, GST-p2 and GST-p3 were each used to raise immune responses in two rabbits as described. Rabbit immune responses were monitored by dot-blot assay using dilute serum to detect affinity purified GST-fusion proteins immobilised on nitrocellulose (Figure 15). This screening indicated the production of antibodies in one of six rabbits (UCR-2) immunised with intracellular domains of receptor P subunits (Figure 15). None of the six rabbits showed immunoreactivity toward GST-fusion proteins before immunisation and one rabbit (UCR-3) failed to develop a significant immune response even after repeated immunisation with GST-p2 (Figure 16). All other rabbits developed a low level immune response in test bleed 1, taken at day 35 post immunisation. This response did not increase significantly by day 56 post immunisation (eg. UCR-1) and was therefore not deemed suitable for further study (Figure 15).

Figure 15

Immune response in rabbit UCR-2 immunised with GST-pl